Received: 22 January 2003Revised: 2 June 2003Accepted: 4 June 2003Published online: 17 July 2003© Springer-Verlag 2003

Abstract Background: In the indus-trialized world age-related maculardegeneration (ARMD) is the leadingcause for legal blindness beyond theage of 65 years. Recent studies indi-cate that the density of the macularpigment (MP) may play a centralrole in the development and progres-sion of age-related maculopathy(ARM). We present results of mea-surements of macular pigment densi-ty (MPD) with a modified confocalscanning laser ophthalmoscope andcompare macular pigment densityvalues obtained from reflectance andautofluorescence images in healthysubjects. Methods: A modified con-focal scanning laser ophthalmoscope(Heidelberg Engineering, Heidel-berg) was optimised for fundus re-flectance and autofluorescence imag-es at 488 nm and 514 nm. For auto-fluorescence imaging a band passbarrier filter with a short wavelengthcut off at 530 nm was used. MPDmaps were obtained, and MPD was

evaluated within two degrees aroundthe foveal center. In this study we in-cluded 109 healthy subjects agedfrom 18–75 years. Results: AverageMPD ranged between 0.16 densityunits (D.U.) and 0.22 D.U. depend-ing on the techniques used for themeasurement. Using the reflectancemethod the mean MPD was0.16±0.06 D.U., whereas a MPD of0.22±0.07 D.U. resulted from auto-fluorescence images with the 530-nm barrier filter. Conclusions: Thisstudy demonstrates the good feasi-bility of MPD measurements with amodified confocal SLO. In agree-ment with previous studies we foundhigher macular pigment density val-ues calculated from autofluorescenceimages than from reflectance imag-es. We could not find any differencesin MPD related to age and gender.Prospective longitudinal studies toverify the influence of MPD on thedevelopment and progression ofARM are needed.

Graefe’s Arch Clin Exp Ophthalmol(2003) 241:647–651

DOI 10.1007/s00417-003-0730-9

C L I N I C A L I N V E S T I G AT I O N

Henrike WüstemeyerAndreas MoessnerCornelia JahnSebastian Wolf

Macular pigment density in healthy subjectsquantified with a modified confocal scanninglaser ophthalmoscope

Introduction

Age-related macular degeneration (ARMD) is the lead-ing cause of legal blindness in patients over 65 years ofage in the industrialized world [9, 10]. The efforts to findmechanisms of disease-onset on the one hand and mech-anisms for effective therapy on the other are manifold[15, 16, 21, 28]. The third effort is research on effectivepreventive schemes. Macular pigment (MP), which con-sists of the carotenoids lutein and zeaxanthin, has antiox-idant properties as well as the ability to filter short-wave-

length blue light [8, 18]. Free radicals and short-wave-length blue light may both cause damage in the fovealregion, and therefore their involvement in the develop-ment of ARMD is discussed [1]. In this context MP maybe discussed as playing a role in the development ofARM and ARMD [2]. High macular pigment density(MPD) may be protective in the development of ARMD[19, 22]. Before the features of dry ARMD with geo-graphic atrophy or exudative ARMD with neovascularmembranes can be diagnosed on the fundus, the intactbalance within the retina, the retinal pigment epithelium

The authors have no proprietary interest inthis study, which was facilitated by grantno. DFG Wo 478/11 and was presented inpart at the annual meeting of the DeutscheOphthalmologische Gesellschaft (DOG),Berlin, 2002.

Supported by DFG: Wo 478/11

H. Wüstemeyer · A. Moessner · C. JahnS. Wolf (✉)Klinik und Poliklinik für Augenheilkunde,Universität Leipzig,Liebigstrasse 10–14, 04103 Leipzig, Germanye-mail: wolfs@medizin.uni-leipzig.deTel.: +49-341-9721501Fax: +49-341-9721509

(RPE) and the choroid is already deeply disturbed, lead-ing to disease precursors. Drusen and pigmentary chang-es that define the age-related maculopathy (ARM) pre-cede ARMD and represent advanced steps in the under-lying causes leading to the disease [6]. We have intro-duced an instrument to quantify MPD by means of amodified confocal scanning laser ophthalmoscope (SLOHeidelberg Engineering, Heidelberg) [27]. It is our aimto find out whether MPD can be used as a diagnostic pa-rameter in order to assess the integrity of the macula andthe risk to progress from low MPD to ARM and ARMD,respectively.

Subjects and methods

Reflectometry and autofluorescence functional imaging

For quantification of MPD we used a modified confocal SLO. It isoptimised to record reflectance images at 488 nm and 514 nmwavelengths. We obtained high-resolution pictures at 488 nm and514 nm wavelengths with an argon laser. For easy assessment ofMPD we developed a program to create density maps (Fig. 1). Adensity map or functional image is created by digital subtractionof the log reflectance images. The difference in absorption by pig-ments other than MP (e.g., retinal and choroidal blood, visual pig-ments, RPE and choroidal melanin) between the fovea and 5° out-side the fovea can be neglected. Since the absorption of macularpigment at 488 nm wavelength is high and at 514 nm wavelengthclose to zero, we can determine MPD by comparing foveal andparafoveal reflectance at 488 nm and 514 nm. We used a reflec-tance method (REF) as well as an autofluorescence (AF) methodfor quantification of MPD (Fig. 2). For AF imaging we used aband pass filter with a short wavelength cut-off at 530 nm. For as-sessment of MPD, autofluorescence images were processed as de-

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Fig. 1 Examples of REF andAF functional maps and modesof analysis. Upper left: REFfunctional map. Lower left: AFfunctional map. Upper right:MPD distribution including pe-ripheral and central MPD in atest field of 1° height, centeredon the fovea. Lower right:pseudo-topographic map ofMPD. Grey values representingMPD are transformed intograded altitude (test field16×14). Macular pigment den-sity appears as central peak

Fig. 2 Scheme of autofluorescence (AF) and reflectance (REF)measurements. Above: MP absorbance spectrum (dark curve) withmaximal absorbance at 460 nm. Excitation at 488 and 514 nm,band pass filter at 530 nm, detection of lipofuscin autofluores-cence with a maximum at 620–630 nm wavelengths. Below: MPabsorbance spectrum (dark curve) with maximal absorbance at460 nm. Excitation and detection of reflectance at 488 and 514 nmwavelengths

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scribed above. Further details concerning the analytic procedurehave been published and are described elsewhere [13, 27].

Subjects

We recruited 109 healthy subjects (52 males, 57 females) agedfrom 16 to 76 years (mean 46±18 years). Fifty-four subjects wereyounger, 55 subjects were older than 50 years of age. The age dis-tribution had two peaks, one at the age of 25 years, the other oneat the age of 62 years (Fig. 3). Subjects were in good generalhealth, and retinal pathology was not present in the examined eye.Visual acuity was 20/20 (ETDRS visual acuity chart). The studyfollowed the tenets of the Declaration of Helsinki and was ap-proved by the local ethics committee.

Examination

All subjects underwent a comprehensive ocular examination. Thepupil of the test eye was dilated with eye drops containing 0.5%tropicamide and 2.5% phenylephrine. Macular pigment measure-ments were obtained with the modified SLO. The patients werepositioned in front of the SLO and instructed to look straight andsteady. After obtaining good fixation and focusing the 20° field ofthe scanning laser ophthalmoscope on the macular region imagesat 488 nm and 514 nm wavelengths with the REF mode as well asAF mode were taken. MPD maps were processed afterwards, andmean MPD was calculated for a 2°-diameter circle centered on thefovea. For statistical analysis we used a paired t-test to compareMPD quantified with both the REF and AF methods. For correla-tion of age and MPD we used a correlation z-test. For comparisonof groups related to age or gender we used an unpaired t-test.

Results

Macular pigment density was calculated from REF andAF functional maps. Mean MPD of 109 healthy subjectswas MPDREF=0.155±0.06 density units (D.U.) for theREF method and MPDAF=0.215±0.07 D.U. for the AFmethod. AF imaging leads to statistically significanthigher mean MPD values than REF imaging (P<0.01).Comparing the group of subjects younger than 50 yearsof age (n=54) with the one older than 50 years (n=55),

we obtained a mean MPDREF=0.179±0.05 D.U.(<50 years) and MPDREF=0.132±0.06 D.U. (>50 years)for REF images, and a mean of MPDAF=0.215±0.06 D.U. (<50 years) and MPDAF=0.214±0.08 D.U.(>50 years) for AF images (Fig. 4). Using the REF

Fig. 3 Age distribution of the 109 examined subjects. Distributionhas two peaks at 25 and 61 years. Both subgroups follow Gaussiandistribution

Fig. 4 Above: correlation of MPD and age. AF values (grey spots)and regression show no age relation for MPD (R2=0.001); REFvalues (black spots) show a decline of MPD with age (R2=0.196).Below: Difference in MPD in age groups over and under 50 years.A significant difference appears in REF measurements

Fig. 5 Box plot on MPD in male and female subjects measuredwith REF and AF images. No statistically significant differenceoccurs

mode, MPD is significantly higher in subjects aged lessthan 50 years (P<0.01), whereas there is no such differ-ence in the AF mode (P=0.93). Testing the correlation ofMPD to age, we get a decline of MPD with age in theREF method (Y=0.218; R2=0.196; P<0.01), whereas us-ing AF images for quantification, there is no age-relatedchange in MPD (Y=0.219; R2=0.001; P=0.78) (Fig. 4).Comparing the MPD in male (n=52) and female (n=57)subjects, we found no significant difference betweenboth groups, neither in the REF nor in the AF method.Based on REF images the mean MPD for female sub-jects is MPDREF=0.161±0.05 D.U. and MPDAF=0.217±0.07 D.U. for AF images. In males, the results wereMPDREF=0.149±0.06 D.U. for REF and MPDAF=0.212±0.07 D.U. for AF images (Fig. 5).

Discussion

The analysis of risk factors linked to the development ofARM and ARMD has come progressively more into fo-cus in epidemiological studies [11, 17, 20, 25]. The roleof macular pigment as a predictive factor in the develop-ment of ARMD in longitudinal measurements is yet tobe determined. MPD has been measured by means ofdifferent devices ranging from in vitro measurementsand subjective methods to objective measurements [1, 3,5, 23, 24, 26]. Delori et al. have developed a concept forquantifying MPD objectively based on autofluorescenceimages and compared the results of this with reflectome-try and heterochromatic flicker photometry [12, 13].

We have examined a group of 109 healthy subjectsand obtained functional maps based on reflectance(REF) and autofluorescence (AF) images. We then quan-tified mean macular pigment density (MPD) in a 2-de-gree area centered on the fovea. The results were ana-lyzed under various aspects such as age, gender and de-pendency on the smoking habit. Comparing results ob-tained from REF and AF functional maps, it is evidentthat the AF method provides higher mean MPD valuesthan the REF method. This finding is consistent with thefindings of Delori et al. [13]. The effect may be due toreflectance and/or absorption artifacts caused by the lensand the inner limiting membrane (ILM). The fact that theeffect is even more pronounced in the group of subjectsolder than 50 years of age indicates that these disturbingfactors increase with age-related lens and ILM changes.Mathematically, the reflectance method is based on theassumption that 100% of the applied light is reflected af-ter passing the macular pigment. If part of the light is re-flected before, this amount of light is not affected by theabsorbency of macular pigment and leads to false-lowvalues for MPD. The autofluorescence on the contrary isbased on the detection of lipofuscin autofluorescence af-ter excitation with defined wavelengths other than theautofluorescence wavelengths (Fig. 1). It is therefore less

susceptible for reflectance artifacts. The AF methodseems to be more reliable in terms of quantification.Checking on the age dependency of MPD, we split theexamined subjects into two groups. One group wasyounger, the second group was older than 50 years ofage. For the REF method we found a statistically signifi-cant difference in MPD with the younger age group hav-ing higher MPD values than subjects older than 50 years.In the AF method this could not be confirmed. Here, weonly found a higher scatter of the MPD in older subjects.Testing the correlation of age and MPD we found a cor-relation of age and MPD of 19.6% (R2=0.196) for theREF method and 0.1% (R2=0.001) for AF measure-ments. The discrepancy between the methods has beendiscussed above. The higher susceptibility of the REFmethod to reflectance artifacts may also be responsiblefor a feigned age-dependency of MPD. An increasedlens opacity and increased reflection from the ILM mayhave a higher impact in older subjects than in the young.In autofluorescence we did not find an age-related de-cline in MPD, but a greater interindividual MPD varietyin subjects over 50 years of age. Some studies show dif-ferent results indicating that increased age leads to a de-cline in MPD [2, 14]. Hammond et al. have measuredMPD with a psychophysical method using flicker pho-tometry. For the decline with age they found r=−0.14,meaning that 1.9% (R2=0.019) of the decrease in MPD isage related. We see our results obtained with the AFmethod as being in agreement with the results of Ham-mond et al., as both studies show no statistically signifi-cant decline in MPD with age. Former studies confirmour results and also did not find any age-related declinein MPD [7]. There was no evident gender-related differ-ence in MPD in either the REF or in AF measurements.This is in agreement with findings of other groups [2, 4,13]. The same is true for the impact of smoking onMPD, although our group of smokers was too small fordefinite conclusions.

For further studies on patients with ARM we preferautofluorescence-based quantification of macular pig-ment density. Longitudinal studies that follow up MPDin high-risk eyes for ARMD are needed to clinically as-sess the predictive ability of MPD measurements. It isnecessary to compare healthy subjects’ MPD to the MPDof patients with different stages of ARM. The number ofdrusen, their size and distribution may affect the MPD.The follow-up of these patients will reveal the ability ofthe MPD measurement to assess the course of the dis-ease or to assess the risk of progression to late stages ofARMD.

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